Methods and compositions for identifying modulators of G-protein-coupled receptors

Abstract

The present invention provides methods and compositions that can be used to identify modulators of G-protein-coupled receptors.

What is claimed is:  
     
       1. A method for identifying a non-Gq G-protein-coupled receptor (GPCR) modulator, comprising the steps of: 
       (i) contacting a candidate non-Gq GPCR modulator with a host cell comprising a non-Gq GPCR and a mutant q-type G protein wherein said mutant comprises an alteration in one or more of the first 10 N-terminal amino acids of the α q  subunit and wherein said mutant is responsive to at least one non-q-type GPCR in said host cell; and  
       (ii) assaying for a q-type G protein response.  
     
     
       2. The method according to  claim 1 , wherein said cell comprises a recombinant polynucleotide encoding a non-q-type GPCR. 
     
     
       3. The method according to  claim 1 , wherein said cell has been transformed with a vector encoding the mutant q-type protein. 
     
     
       4. The method according to  claim 1 , wherein assaying for a q-type G protein response comprises assaying for activation of phospholipase C. 
     
     
       5. The method according to  claim 1 , wherein assaying for a q-type G protein response comprises assaying for hydrolysis of phosphatidylinositols. 
     
     
       6. The method according to  claim 1 , wherein assaying for a q-type G protein response comprises assaying for calcium mobilization. 
     
     
       7. The method according to  claim 1 , wherein said modulator is a GPCR agonist that binds to and activates a non-q-type GPCR. 
     
     
       8. A method according to  claim 7 , wherein said modulator is a GPCR agonist that binds to and activates a Gi-type GPCR. 
     
     
       9. A method according to  claim 7 , wherein said modulator is a GPCR agonist that binds to and activates a Gs-type GPCR. 
     
     
       10. The method according to  claim 1 , wherein said modulator is a GPCR antagonist that binds to and competitively inhibits a non-q-type GPCR. 
     
     
       11. The method according to  claim 10 , wherein said method comprises the additional step of contacting said host cell with an agonist of said non-q-type GPCR. 
     
     
       12. The method according to  claim 1 , wherein said modulator is a GPCR antagonist that binds to and non-competitively inhibits a non-q-type GPCR. 
     
     
       13. The method according to  claim 1 , wherein said mutant q-type G-protein transduces the signal of a Gi or Gs type GPCR. 
     
     
       14. The method according to  claim 1 , wherein said alteration is selected from the group consisting of a deletion, substitution and insertion. 
     
     
       15. The method according to  claim 1 , wherein said alteration maintains the alpha-helical nature of the N-terminal region. 
     
     
       16. The method according to  claim 1 , wherein said alteration comprises a substitution of at least one of the first six N-terminal amino acids of the α q  subunit. 
     
     
       17. The method according to  claim 1 , wherein said alteration comprises a substitution of at least two of the first six N-terminal amino acids of the α q  subunit. 
     
     
       18. The method according to  claim 1 , wherein said alteration is a deletion of at least one of the first six N-terminal amino acids of the α q  subunit. 
     
     
       19. The method according to  claim 1 , wherein said alteration is a deletion of at least two of the first six N-terminal amino acids of the α q  subunit. 
     
     
       20. The method according to  claim 1 , wherein said alteration is a deletion of the first six N-terminal amino acids of the α q  subunit. 
     
     
       21. The method according to  claim 1 , wherein said mutant further comprises a substitution of at least one of N-terminal amino acids 7-10 of the α q  subunit. 
     
     
       22. The method according to  claim 21 , wherein said substitution comprises a substitution of at least one of N-terminal amino acids 7, 8, 9 or 10 with an amino acid found at position 1, 2, 3 or 4 of a non-q-type α subunit. 
     
     
       23. The method according to  claim 1 , wherein said mutant further comprises a C-terminal mutation in which one or more amino acids at the C-terminus of the α q  subunit is replaced with one or more amino acids from the C-terminus of a non-q-type α subunit. 
     
     
       24. A method for identifying a non-Gq G-protein-coupled receptor (GPCR) modulator, comprising the steps of: 
       (i) contacting a candidate non-Gq GPCR modulator with a non-Gq GPCR and a mutant q-type G protein wherein said mutant comprises an alteration in one or more of the first 10 N-terminal amino acids of the α q  subunit and wherein said mutant is responsive to said non-q-type GPCR; and  
       (ii) assaying for a q-type G protein response.  
     
     
       25. The method according to  claim 24 , wherein assaying for a q-type G protein response comprises assaying for activation of phospholipase C. 
     
     
       26. The method according to  claim 24 , wherein assaying for a q-type G protein response comprises assaying for hydrolysis of phosphatidylinositols. 
     
     
       27. The method according to  claim 24 , wherein assaying for a q-type G protein response comprises assaying for calcium mobilization. 
     
     
       28. The method according to  claim 24 , wherein said modulator is a GPCR agonist that binds to and activates a non-q-type GPCR. 
     
     
       29. A method according to  claim 24 , wherein said modulator is a GPCR agonist that binds to and activates a Gi-type GPCR. 
     
     
       30. A method according to  claim 24 , wherein said modulator is a GPCR agonist that binds to and activates a Gs-type GPCR. 
     
     
       31. The method according to  claim 24 , wherein said modulator is a GPCR agonist that binds to and competitively inhibits a non-q-type GPCR. 
     
     
       32. The method according to  claim 31 , wherein said method comprises the additional step of contacting said non-Gq GPCR with an agonist of said non-q-type GPCR. 
     
     
       33. The method according to  claim 24 , wherein said modulator is a GPCR agonist that binds to and non-competitively inhibits a non-q-type GPCR. 
     
     
       34. The method according to  claim 24 , wherein said mutant q-type G-protein transduces the signal of a Gi or Gs type GPCR. 
     
     
       35. The method according to  claim 24 , wherein said alteration is selected from the group consisting of a deletion, substitution and insertion. 
     
     
       36. The method according to  claim 24 , wherein said alteration maintains the nature of the N-terminal region. 
     
     
       37. The method according to  claim 24 , wherein said alteration comprises a of at least one of the first six N-terminal amino acids of the α q  subunit. 
     
     
       38. The method according to  claim 24 , wherein said alteration comprises a substitution of at least two of the first six N-terminal amino acids of the α q  subunit. 
     
     
       39. The method according to  claim 24 , wherein said alteration is a deletion of at least one of the first six N-terminal amino acids of the α q  subunit. 
     
     
       40. The method according to  claim 24 , wherein said alteration is a deletion of at least two of the first six N-terminal amino acids of the α q  subunit. 
     
     
       41. The method according to  claim 24 , wherein said alteration is a deletion of the first six N-terminal amino acids of the α q  subunit. 
     
     
       42. The method according to  claim 24 , wherein said mutant further comprises a substitution of at least one of N-terminal amino acids 7-10 of the α q  subunit. 
     
     
       43. The method according to  claim 42 , wherein said substitution comprises a substitution of at least one of N-terminal amino acids 7, 8, 9 or 10 with an amino acid found at position 1, 2, 3 or 4 of a non-q-type α subunit. 
     
     
       44. The method according to  claim 24 , wherein said mutant further comprises a C-terminal mutation in which one or more amino acids at the C-terminus of the α q  subunit is replaced with one or more amino acids from the C-terminus of a non-q-type α subunit. 
     
     
       45. A recombinant polynucleotide encoding a mutant q-type G protein wherein said mutant comprises a first alteration in one or more of the first 6 N-terminal amino acids of q-type G protein and a substitution of any one of N-terminal amino acids 7-10 of said q-type G protein, wherein said mutant is responsive to at least one non-q-type GPCR. 
     
     
       46. The recombinant polynucleotide according to  claim 45 , wherein said alteration is selected from the group consisting of a deletion, substitution and insertion. 
     
     
       47. The recombinant polynucleotide according to  claim 45 , wherein said alteration maintains the alpha-helical nature of the N-terminal region. 
     
     
       48. The recombinant polynucleotide according to  claim 45 , wherein said first alteration comprises a substitution of at least one of the first six N-terminal amino acids of the α q  subunit. 
     
     
       49. The recombinant polynucleotide according to  claim 45 , wherein said first alteration comprises a substitution of at least two of the first six N-terminal amino acids of the α q  subunit. 
     
     
       50. The recombinant polynucleotide according to  claim 45 , wherein said first alteration is a deletion of at least one of the first six N-terminal amino acids of the α q  subunit. 
     
     
       51. The recombinant polynucleotide according to  claim 45 , wherein said first alteration is a deletion of at least two of the first six N-terminal amino acids of the α q  subunit. 
     
     
       52. The recombinant polynucleotide according to  claim 45 , wherein said first alteration is a deletion of the first six N-terminal amino acids of the α q  subunit. 
     
     
       53. The recombinant polynucleotide according to  claim 45 , wherein said substitution of any one of N-terminal amino acids 7-10 comprises a substitution of at least one of amino acids 7-10 of the α q  subunit. 
     
     
       54. The recombinant polynucleotide according to  claim 45 , wherein said substitution of any one of N-terminal amino acids 7-10 comprises a substitution of at least one of amino acids 7, 8, 9 or 10 with amino acids found at positions 1, 2, 3 or 4 of a non-q-type α subunit. 
     
     
       55. The recombinant polynucleotide according to  claim 45 , further comprising a C-terminal mutation in which one or more amino acids at the C-terminus of the α q  subunit is replaced with an amino acid from the C-terminus of a non-q-type α subunit. 
     
     
       56. A host cell transformed with a recombinant polynucleotide according to  claim 45 . 
     
     
       57. The cell of  claim 56 , further transformed with a recombinant polynucleotide encoding a non-q-type GPCR. 
     
     
       58. The cell of  claim 57 , wherein said non-q-type GPCR is a Gi-type GPCR. 
     
     
       59. The cell of  claim 57 , wherein said non-q-type GPCR is a Gs-type GPCR. 
     
     
       60. The recombinant polynucleotide of  claim 25  further comprising an additional alteration in the C-terminal region of said q-type G protein, wherein said mutant is responsive to at least one non-q-type GPCR. 
     
     
       61. The recombinant polynucleotide according to  claim 60 , wherein said mutant further comprises a C-terminal mutation in which one or more amino acids at the C-terminus of the α q  subunit is replaced with one or more amino acids from the C-terminus of a non-q-type α subunit. 
     
     
       62. A recombinant polynucleotide encoding a mutant q-type G protein wherein said mutant comprises a first alteration in one or more of the first 6 N-terminal amino acids of q-type G protein and a second alteration in the C-terminal region of said q-type G protein, wherein said mutant is responsive to at least one non-q-type GPCR. 
     
     
       63. The recombinant polynucleotide according to  claim 62 , wherein said alteration is selected from the group consisting of a deletion, substitution and insertion. 
     
     
       64. The recombinant polynucleotide according to  claim 62 , wherein said alteration maintains the alpha-helical nature of the N-terminal region. 
     
     
       65. The recombinant polynucleotide according to  claim 62 , wherein said first alteration comprises a substitution of at least one of the first six amino acids of the α q  subunit. 
     
     
       66. The recombinant polynucleotide according to  claim 62 , wherein said first alteration comprises a substitution of at least two of the first six amino acids of the α q  subunit. 
     
     
       67. The recombinant polynucleotide according to  claim 62 , wherein said first alteration is a deletion of at least one of the first six amino acids of the α q  subunit. 
     
     
       68. The recombinant polynucleotide according to  claim 62 , wherein said first alteration is a deletion of at least two of the first six N-terminal amino acids of the α q  subunit. 
     
     
       69. The recombinant polynucleotide according to  claim 62 , wherein said first alteration is a deletion of the first six N-terminal amino acids of the α q  subunit. 
     
     
       70. The recombinant polynucleotide according to  claim 62 , wherein said mutant further comprises a C-terminal mutation in which one or more amino acids at the C-terminus of the α q  subunit is replaced with one or more amino acids from the C-terminus of a non-q-type α subunit. 
     
     
       71. A host cell transformed with a recombinant polynucleotide according to  claim 62 . 
     
     
       72. The cell of  claim 71 , further transformed with a recombinant polynucleotide encoding a non-q-type GPCR. 
     
     
       73. The cell of  claim 72 , wherein said non-q-type GPCR is a Gi-type GPCR. 
     
     
       74. The cell of  claim 72 , wherein said non-q-type GPCR is a Gs-type GPCR. 
     
     
       75. A recombinant polynucleotide encoding a mutant q-type G protein wherein said mutant comprises a substitution of at least one of the first six N-terminal amino acids of a q-type G protein, wherein said mutant is responsive to at least one non-q-type GPCR. 
     
     
       76. The recombinant polynucleotide of  claim 75  further comprising an additional alteration in the C-terminal region of said q-type G protein, wherein said mutant is responsive to at least one non-q-type GPCR. 
     
     
       77. A recombinant polynucleotide encoding a mutant q-type G protein wherein said mutant comprises a substitution of at least two of the first six N-terminal amino acids of a q-type G protein, wherein said mutant is responsive to at least one non-q-type GPCR. 
     
     
       78. The recombinant polynucleotide of  claim 77  further comprising an additional alteration in the C-terminal region of said q-type G protein, wherein said mutant is responsive to at least one non-q-type GPCR.